A Cytochrome P450 Conserved in Insects Is Involved in Cuticle Formation

The sequencing of numerous insect genomes has revealed dynamic changes in the number and identity of cytochrome P450 genes in different insects. In the evolutionary sense, the rapid birth and death of many P450 genes is observed, with only a small number of P450 genes showing orthology between insects with sequenced genomes. It is likely that these conserved P450s function in conserved pathways. In this study, we demonstrate the P450 gene, Cyp301a1, present in all insect genomes sequenced to date, affects the formation of the adult cuticle in Drosophila melanogaster. A Cyp301a1 piggyBac insertion mutant and RNAi of Cyp301a1 both show a similar cuticle malformation phenotype, which can be reduced by 20-hydroxyecdysone, suggesting that Cyp301a1 is an important gene involved in the formation of the adult cuticle and may be involved in ecdysone regulation in this tissue

Transcriptome Analysis of Drosophila melanogaster Third Instar Larval Ring Glands Points to Novel Functions and Uncovers a Cytochrome p450 Required for Development

In Drosophila melanogaster larvae, the ring gland (RG) is a control center that orchestrates major developmental transitions. It is a composite organ, consisting of the prothoracic gland, the corpus allatum, and the corpora cardiaca, each of which synthesizes and secretes a different hormone. Until now, the RG’s broader developmental roles beyond endocrine secretion have not been explored. RNA sequencing and analysis of a new transcriptome resource from D. melanogaster wandering third instar larval RGs has provided a fascinating insight into the diversity of developmental signaling in this organ. We have found strong enrichment of expression of two gene pathways not previously associated with the RG: immune response and fatty acid metabolism. We have also uncovered strong expression for many uncharacterized genes. Additionally, RNA interference against RG-enriched cytochrome p450s Cyp6u1 and Cyp6g2 produced a lethal ecdysone deficiency and a juvenile hormone deficiency, respectively, flagging a critical role for these genes in hormone synthesis. This transcriptome provides a valuable new resource for investigation of roles played by the RG in governing insect development

Evolutionary changes in gene expression, coding sequence and copy-number at the Cyp6g1 locus contribute to resistance to multiple insecticides in Drosophila

Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster-D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species

Evolutionary changes in gene expression, coding sequence and copy-number at the Cyp6g1 locus contribute to resistance to multiple insecticides in Drosophila

Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster–D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species

Cellular rescue in a zebrafish model of congenital muscular dystrophy type 1A

Laminins comprise structural components of basement membranes, critical in the regulation of differentiation, survival and migration of a diverse range of cell types, including skeletal muscle. Mutations in one muscle enriched Laminin isoform, Laminin alpha2 (Lama2), results in the most common form of congenital muscular dystrophy, congenital muscular dystrophy type 1A (MDC1A). However, the exact cellular mechanism by which Laminin loss results in the pathological spectrum associated with MDC1A remains elusive. Here we show, via live tracking of individual muscle fibres, that dystrophic myofibres in the zebrafish model of MDC1A maintain sarcolemmal integrity and undergo dynamic remodelling behaviours post detachment, including focal sarcolemmal reattachment, cell extension and hyper-fusion with surrounding myoblasts. These observations imply the existence of a window of therapeutic opportunity, where detached cells may be "re-functionalised" prior to their delayed entry into the cell death program, a process we show can be achieved by muscle specific or systemic Laminin delivery. We further reveal that Laminin also acts as a pro-regenerative factor that stimulates muscle stem cell-mediated repair in lama2-deficient animals in vivo. The potential multi-mode of action of Laminin replacement therapy suggests it may provide a potent therapeutic axis for the treatment for MDC1A

Overexpression of different <i>Cyp6g1</i> orthologs confers resistance to different insecticides.

<p>A) Changes in survival following exposure to three classes of insecticide by expression of Cyp6g1 orthologs from D. melanogaster, D. simulans, D. willistoni and D. virilis in a consistent genetic background. Resistance ratio (RR) was calculated by comparing the concentration of insecticide that killed 50% of insects (the LC₅₀) between the line expressing the ortholog and the background strain, 86Fb, which was genetically identical except for the absence of the Cyp6g1 construct. Results marked with an asterisk were statistically significant (p&lt;0.05). Orthologs from D. melanogaster and D. simulans were functionally identical at a qualitative level, both providing resistance to all three chemicals, but the resistance profile varied between the other three orthologs. B) Comparison of the potential of the CYP6G1 orthologs to cause resistance when overexpressed. D. melanogaster and D. simulans orthologs cause resistance to a range of chemicals, whilst the ortholog from D. willistoni and the two paralogs from D. virilis only conferred resistance to one of the chemicals tested. These results suggest that adaptation of the protein has occurred repeatedly in Drosophila. The scale bar indicates the number of substitutions per four-fold degenerate site in the genomes of the species (inferred from Stark et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084879#pone.0084879-Stark1" target="_blank">[21]</a>).</p

Unrooted neighbour-joining tree of predicted CYP6G1 amino acid sequences from twelve Drosophila species.

<p>The node labels show bootstrap values from 1000 iterations. Paralogs labelled in the same colour are from the same species. The clustering of paralogs from the same species rather than of orthologs between species supports the hypothesis that the duplications and triplications occurred independently in the separate lineages.</p

Cyp6g1 and Cyp6g2 copy number in twelve Drosophila species.

<p>Cyp6g1 is duplicated in D. willistoni, D. grimshawi and some strains of D. melanogster, and triplicated in D. virilis. The third copy of <i>Cyp6g1</i> in the strain of <i>D. virilis</i> used for this study has an inactivating mutation, but this mutation is not present in the sequenced strain, so it is not formally a pseudogene. Comparison with the phylogeny of the species suggests that multiple independent duplication events occurred (cladogram inferred from Stark et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084879#pone.0084879-Stark1" target="_blank">[21]</a>). In contrast, Cyp6g2 has 1∶1 orthologs in all twelve Drosophila species analyzed.</p